Embryonic stem cells (ES cells) were first cultured from mouse embryos using a feeder layer of mouse fibroblasts or media conditioned with buffalo rat liver cells. The established ESC lines from mouse embryos have a characteristic phenotype consisting of a large nucleus, a prominent nucleolus, and relatively little cytoplasm. Such cells can be grown relatively indefinitely using the appropriate culture conditions. They can be induced to differentiate in vitro using retinoic acid or spontaneously by removal of the feeder layer or conditioned media. In addition, these cells can be injected into a mouse blastocyst to form a somatic and germ line chimera. This latter property has allowed mouse ESCs to be used for the production of transgenic mice with specific changes to the genome. See M. Evans et al., Nature 292, 154 (1981); G. Martin, Proc. Natl. Acad. Sci. USA 78, 7638 (1981); A. Smith et al., Developmental Biology 121, 1 (1987); T. Doetschman et al., Developmental Biology 127, 224 (1988)(; A. Handyside et al., Roux's Arch Dev. Biol. 198, 48 (1989).
The active compound that allows the culture of murine embryonic stem cells has been identified as differentiation inhibiting activity (DIA), also known as leukemia inhibitory factor (LIF). See A. Smith, J. Tiss. cult. Meth. 13, 89 (1991); J. Nichols et al., Development 110, 1341 (1990). Recombinant forms of LIF can be used to obtain ESCs from mouse embryos. See S. Pease et al., Developmental Biology 141, 344 (1990). Also see U.S. Pat. No. 5,166,065 issued Nov. 24, 1992 to Williams, et al.
Subsequent to the work with mouse embryos, several groups have attempted to develop stem cell lines from sheep, pig and cattle. A few reports indicate that a cell line with a stem cell-like appearance has been cultured from porcine embryos using culture conditions similar to that used for the mouse. See M. Evans et al., PCT Application W090/03432; E. Notarianni et al., J. Reprod. Fert., Suppl. 41, 51 (1990); J. Piedrahita et al., Theriogenology 34, 879 (1990); E. Notarianni et al., Proceedings of the 4th World Congress on Genetics Applied to Livestock Productions, 58 (Edinburgh, July 1990).
Attempts have been made regarding the culture of embryonic stem cells from avian embryos. It is difficult to establish a continuous line of chicken cells without viral or chemical transformation, and most primary chicken lines do not survive beyond 2-3 months. The culture of cells from the unincubated embryo is difficult, and under reported conditions such cells do not survive beyond two weeks. See E. Mitrani et al., Differentiation 21, 56-61 (1982); E. Sanders et al., Cell Tissue Res. 220, 539 (1981).
In U.S. Pat. No. 5,340,740 Petille et al. cultured chicken embryo cells on a mouse feeder layer in the presence of conditioned media and obtained the cultured stem cells.
Embryonic stem (ES) cells, the pluripotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment to contribute normally to all tissues including the germline (for review see Robertson, E. G. (1986) Trends in Genetics 2:9-13). Not only can ES cells propagated in vitro contribute efficiently to the formation of chimeras, including germline chimeras, but in addition, these cells can be manipulated in vitro without losing their capacity to generate germ-line chimeras (Robertson, E. J., et al. (1986) Nature, 323:445-447).
ES cells thus provide a route for the generation of transgenic animals such as transgenic mice, a route which has a number of important advantages compared with more conventional techniques, such as zygote injection and viral infection (Wagner and Stewart (1986) in Experimental Approaches to Embryonic Development. J. Rossant and A. Pedersen eds. Cambridge; Cambridge University Press), for introducing new genetic material into such animals.
However, it is known that ES cells and certain EC (embryonal carcinoma) cell lines will only retain the stem cell phenotype in vitro when cultured on a feeder layer of fibroblasts (such as murine STO cells, e.g., Martin, G. R. and Evans, M. J. (1975) Proc. Natl. Acad. Sci. USA 72:1441-1445) or when cultured in medium conditioned by certain cells (e.g. Koopman, P. and Cotton, R. G. H. (1984) Exp. Cell Res. 154:233-242; Smith, A. G. and Hooper, M. L. (1987) Devel. Biol. 121:1-91). In the absence of feeder cells or conditioned medium, the ES cells spontaneously differentiate into a wide variety of cell types, resembling those found during embryogenesis and in the adult animal. The factors responsible for maintaining the pluripotency of ES cells have, however, remained poorly characterized.
The above methods involve the use of ES cells as starting materials. Very limited numbers of such cells are available. Any method which would allow for producing large numbers of ES cell would be very desirable.